The focus of our research program is to understand the structural basis of muscle contraction at the molecular level. The benefits of the research are not limited to understanding muscle contraction, but the contractile system of muscle can serve as a prototype for many other motile processes in living cells, e.g. cell division and nutrient transport. The basic processes of muscle contraction are well understood: it is a result of cyclic interactions between myosin and actin, driven by the energy of actomyosin ATP hydrolysis. Muscle shortening involves the relative sliding of two sets of filaments: the thick, myosin containing filaments and the thin, actin containing filaments. Force is generated by myosin heads (cross-bridges) interacting cyclically with specific sites along the actin filaments. Since the availability of the crystal structures of the contractile proteins, and with the advent of single molecule assays, the field has made great strides in understanding the underlying processes. However, the details of the mechanism of transduction of chemical to mechanical energy still remains largely unresolved. One of the obstacles is that most of the studies at the molecular level are based on isolated, in vitro systems, e.g. the atomic structure of the myosin head is obtained alone without interacting with actin and EM reconstruction is based on isolated filaments. The link between the information obtained from the in vitro systems and the actual processes occurring intact muscle is still largely missing. The aim of our efforts is to provide such a link. Although crystal structures of myosin reveals ligand-dependent structural differences, to gain full understanding of the contraction mechanism, it is critical to determine the in vivo structures in a fully functioning muscle cell. X-ray diffraction from permeabilized muscle cells, the technique used in the present study, is one of the few techniques that reveal the structures as they occur in functioning muscle cells, albeit at relatively low resolution. We have been systematically studying the distributions and orientations of the myosin cross-bridges in living muscle cells during the ATP hydrolysis cycle. Major characteristics of several the intermediate states have been determined (Ref. 1 & 2). We have also shown that the distribution of myosin heads on the myosin filament surface reflects the conformation of the molecule, which is in turn profoundly affected by temperature and ligand at the active site (Ref 3). A helically ordered filament directly reflects that the myosin heads are in the "closed" conformation as determined by crystallography and are poised to interact with actin for force generation. In addition, myosin with one ligand bound can assume multiple conformations. This finding breaks away from the conventional thinking of "one ligand, one conformation", and may be generalized to other proteins. Recently, we have initiated structural studies of cardiac muscle. We are testing the hypothesis that the degree of weak cross-bridge binding, which has been shown to be obligatory for force generation in skeletal muscle, is modulated by changes in lattice spacing in skinned cardiac muscle and thereby force is consequently modulated.